Various methods of separating and detecting deoxyribonucleic acids (DNA) from liquid biological samples are known in the art. One such technique, electrophoresis, involves the migration of charged electrical species when dissolved, or suspended, in an electrolyte through which an electric current is passed. Various conventional forms of electrophoresis are known including free zone electrophoresis, gel electrophoresis, isoelectric focusing, isotachophoresis, and capillary electrophoresis.
Traditionally, the separation and identification of nucleic acids have been accomplished by gel electrophoresis on polymer gels, such as agarose gels or polyacrylamide gels. Since polymer gels can be prepared in various types and sizes and their porosity can also be varied, polymer gels have a wide application. However, due to the instability over time, irreproducibility in the polymerization processes, the fragile nature of the medium, and post-separation detection, gel electrophoresis is generally not suitable for large-scale and high-speed applications (H.-T. Chang and E. S. Yeung, J. Chromatogr. B (1995) 669, 113).
Capillary electrophoresis (CE) represents a significant improvement in electrophoretic analysis of nucleic acids. For example, because CE is performed in very small diameter tubing (typically 50-100 μm) which results in reduced local Joule heating, electrical fields 10- to 100-fold greater than those used in conventional electrophoretic systems can be applied. This affords very high speed runs, improved resolution, and high reproducibility. Also, CE lends itself to on-column detection means including ultraviolet (UV) spectroscopy, amperometric measurement, conductivity measurement, laser-induced fluorescence detection (LIF) or thermo-optical detection. Additionally, CE is well suited for automation, since it provides convenient on-line injection, detection, and real-time data analysis.
Due to the small size of sample volumes injected in a typical CE analysis, the practical application of CE is highly dependent on sensitive detection systems (J. Skeidsvoll and P. M. Ueland, Analytical Biochemistry, (1995) 231, 359-365). There have been several reports of high sensitivity of DNA analysis by CE-LIF when fluorescent intercalating dyes or intercalators are used. Intercalators are planar molecules which interpose between base pairs in nucleic acids or similar structures. The fluorescence of the intercalators increases several-fold upon binding to DNA or RNA, thereby enabling the detection of small amounts of nucleic acids. (U.S. Pat. No. 5,734,058).
Ethidium Bromide (EB) is the most commonly used DNA intercalator, both in gel and capillary electrophoresis. In addition to its fluorescent properties, EB has been reported to provide high CE resolution of double-stranded DNA (dsDNA) (Id.; H.-T. Chang and E. S. Yeung, supra). High-resolution CE separations achieved with EB have been attributed to a reduction in the electrophoretic mobility of DNA when EB-DNA complex is formed. This mobility reduction increases with increasing molecular weight of the DNA (A. Guttman and N. Cooke, Anal. Chem. (1991) 63, 2038-2042). However, because EB binds relatively weakly to DNA (Kdiss=10−6 M−1), the sensitivity of the DNA detection with EB is low. Generally, the detection limit is about 1 ng of double-stranded DNA (dsDNA) in 1 mm×5 mm band on a gel (U.S. Pat. No. 5,312,921).
Recently, an asymmetric cyanine dye, thiazole orange (TO; 4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-ethylidene]-quinolinium iodide) has been suggested as an alternative to EB (U.S. Pat. No. 5,312,921). Although this dye has a higher affinity to DNA and gives a 10-fold higher sensitivity than EB (Skeidsvoll & Ueland, supra), TO does not provide the same DNA separation performance as EB. For example, while the concentration of EB does not affect DNA separation, the quality of DNA separation is very sensitive to DNA-to-dye ratio when TO is used. It has been reported that DNA peak shape greatly deteriorates when DNA-to-dye ratio falls outside of the 9:1 ratio. Consequently, broad peaks and relatively low peak heights are observed when TO is used. One possible explanation for broad peaks is that TO not only intercalates but also binds to the separated DNA strands. Also, an excess of TO may be destabilizing to the DNA double helix, which results in broad peaks (H. E. Schwartz and K. J. Ulfelder, Anal. Chem. 1992, 64, 1737-1740).
Since the discovery of TO as a nucleic acid dye, several improvements to TO and its trimethine homologs have been developed to provide dyes with tighter binding to DNA and greater water solubility (U.S. Pat. No. 5,321,130 and U.S. Pat. No. 5,312,921). These dyes generally involve a modification to the quinolinium portion of the dye and are fairly expensive.
In summary, some conventional intercalating dyes, such as EB, provide a high DNA resolution but have a low affinity and low DNA detection sensitivity. On the other hand, other conventional intercalating dyes, such as TO, provide a high sensitivity of detection but do not provide desirable resolution. Accordingly, a continued interest exists in developing high resolution/high sensitivity techniques for detecting DNA in a sample, particularly where DNA is present in an extremely low concentration.